Regulation of transcription elongation factors

Abstract

The present invention relates to agents, including siRNA and shRNA molecules, small molecules, antisense strands, and ribozymes that are targeted to transcription elongation factors (TEFs), including CDK9 and CycT1, subunits of P-TEFb, Spt4 and Spt5, subunits of DSIF (DRB Sensitivity-Inducing Factor (DSIF)), and Spt6. The present invention also relates to methods for treating disorders associated with aberrant or unwanted TEF expression or activity, including HIV and disorders characterized by unwanted or aberrant cellular proliferation or differentiation, such as cancer.

What is claimed is:  
     
         1 . An isolated ribonucleic acid (RNA) molecule comprising a first nucleotide sequence of at least 16 nucleotides sufficiently complementary to a target region of a TEF or TEF subunit mRNA sequence to direct target-specific RNA interference (RNAi), and a second nucleotide sequence of at least 16 nucleotides complementary to the first nucleotide sequence.  
     
     
         2 . An isolated ribonucleic acid (RNA) molecule, comprising a sense strand and an antisense strand, wherein the antisense strand has a sequence sufficiently complementary to a target region of a TEF or TEF subunit mRNA sequence to direct target-specific RNA interference (RNAi) and wherein the sense strand has a sequence complementary to the sequence of the antisense strand.  
     
     
         3 . The isolated RNA molecule of  claim 2 , wherein the sense and antisense strands each comprise at least 16 nucleotides.  
     
     
         4 . The isolated RNA molecule of any one of claims  1 - 3 , wherein the TEF mRNA is a P-TEFb mRNA  
     
     
         5 . The isolated RNA molecule of any one of claims  1 - 3 , wherein the TEF subunit RNA is a CycT1 mRNA.  
     
     
         6 . The isolated RNA molecule of  claim 4 , wherein the CycT1 mRNA sequence is set forth as SEQ ID NO: 1.  
     
     
         7 . The isolated RNA molecule of any one of claims  1 - 3 , wherein the TEF subunit mRNA is a CDK9 mRNA.  
     
     
         8 . The isolated RNA molecule of  claim 6 , wherein the CDK9 mRNA sequence is set forth as SEQ ID NO: 2.  
     
     
         9 . The isolated RNA molecule of any one of claims  1 - 3 , wherein the TEF mRNA is a DSFI mRNA.  
     
     
         10 . The isolated RNA molecule of any one of claims  1 - 3 , wherein the TEF subunit mRNA is a Spt5 mRNA.  
     
     
         11 . The isolated RNA molecule of  claim 10 , wherein the Spt5 mRNA sequence is set forth as SEQ ID NO: 7.  
     
     
         12 . The isolated RNAmolecule of  claim 10 , wherein the Spt5 mRNA sequence is set forth as SEQ ID NO: 11.  
     
     
         13 . The isolated RNA molecule of any one of claims  1 - 12 , wherein the first nucleotide sequence is fully complementary to the mRNA sequence.  
     
     
         14 . The isolated RNA molecule of any one of claims  2 - 12 , wherein the antisense strand nucleotide sequence is fully complementary to the mRNA sequence.  
     
     
         15 . The isolated RNA molecule of any one of claims  1  and  4 - 13 , further comprising a loop portion comprising 4-11 nucleotides that connects the two nucleotide sequences.  
     
     
         16 . The isolated RNA molecule of any one of claims  1 ,  4 - 13  and  15 , wherein the first and second nucleotide sequences each comprise 16, 17, 18 or 19 nucleotides.  
     
     
         17 . The isolated RNA molecule of any one of claims  1 ,  4 - 13  and  15 , wherein the first and second nucleotide sequences each consist of 20, 21 or 22 nucleotides.  
     
     
         18 . The isolated RNA molecule of any one of claims  2 - 14 , wherein the sense and antisense strands each comprise 16, 17, 18 or 19 nucleotides.  
     
     
         19 . The isolated RNA molecule of any one of claims  2 - 14 , wherein the sense and antisense strands each consist of 20, 21 or 22 nucleotides.  
     
     
         20 . The isolated RNA molecule of any one of the preceeding claims, wherein the target region of the mRNA sequence is located from 100 to 300 nucleotides downstream of the start of translation of the mRNA.  
     
     
         21 . The isolated RNA molecule of any one of the preceeding claims, wherein the target region of the mRNA sequence is located in a 5′ untranslated region (UTR) or a 3′ UTR of the mRNA.  
     
     
         22 . The isolated RNA molecule of  claim 1 , wherein the first or second nucleotide sequence is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 21, SEQ ID NO: 5 and SEQ ID NO: 23.  
     
     
         23 . The isolated RNA molecule of  claim 1 , wherein the first or second nucleotide sequence is selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 25.  
     
     
         24 . An isolated nucleic acid molecule encoding the RNA molecule of any one of the preceding claims.  
     
     
         25 . A vector comprising the isolated nucleic acid molecule of  claim 24 .  
     
     
         26 . The vector of  claim 25 , which is a viral vector, retroviral vector, expression cassette, or plasmid.  
     
     
         27 . The vector of  claim 25 , further comprising an RNA Polymerase III or RNA Polymerase II promoter.  
     
     
         28 . The vector of  claim 25 , wherein the RNA Polymerase III promoter is the U6 or H1 promoter.  
     
     
         29 . A host cell comprising the nucleic acid molecule of any one of claims  1 - 24 .  
     
     
         30 . A host cell comprising the vector of any one of claims  24 - 28 .  
     
     
         31 . The host cell of  claim 29  or  30 , which is a mammalian host cell.  
     
     
         32 . The host cell of  claim 31 , which is a non-human mammalian cell.  
     
     
         33 . The host cell of  claim 31 , which is a human cell.  
     
     
         34 . A therapeutic composition comprising the nucleic acid molecule of one of claims  1 - 24 , and a pharmaceutically acceptable carrier.  
     
     
         35 . A method of treating a subject having a disorder characterized by aberrant or unwanted cellular proliferation, the method comprising administering to the subject a therapeutically effective amount of the composition of  claim 34 .  
     
     
         36 . A method of treating a subject having a disorder characterized by aberrant or unwanted cellular proliferation, the method comprising administering to the subject a therapeutically effective amount of the nucleic acid molecule of any one of claims  1 - 24 .  
     
     
         37 . The method of  claim 35  or  36 , wherein the therapeutically effective amount is an amount effective to inhibit the expression or activity of a TEF, or subunit thereof.  
     
     
         38 . The method of  claim 37 , wherein the TEF is P-TEFb  
     
     
         39 . The method of  claim 37 , wherein the TEF is DSIF.  
     
     
         40 . The method of  claim 37 , wherein the TEF subunit is selected from the group consisting of CycT1, CDK9 and Spt5.  
     
     
         41 . The method of any one of claims  35 - 40 , wherein the disorder is cancer.  
     
     
         42 . The method of  claim 41 , wherein the cancer is selected from the groups consisting of carcinomas, sarcomas, metastatic disorders and hematopoietic neoplastic disorders.  
     
     
         43 . The method of  claim 42 , wherein the hematopoietic neoplastic disorder is a leukemia.  
     
     
         44 . A method of treating a subject infected with HIV, the method comprising administering to the subject a therapeutically effective amount of the composition of  claim 34 .  
     
     
         45 . A method of treating a subject infected with HIV, the method comprising administering to the subject a therapeutically effective amount of the nucleic acid molecule of any one of claims  1 - 24 .  
     
     
         46 . The method of  claim 44  or  45 , wherein the therapeutically effective amount is an amount effective to inhibit the expression or activity of a TEF, or subunit thereof.  
     
     
         47 . The method of  claim 46 , wherein the TEF is P-TEFb  
     
     
         48 . The method of  claim 46 , wherein the TEF is DSIF.  
     
     
         49 . The method of  claim 46 , wherein the TEF subunit is selected from the group consisting of CycT1, CDK9 and Spt5.  
     
     
         50 . A method of treating a subject having a disorder characterized by aberrant or unwanted expression of a gene whose expression is regulated by P-TEFb, the method comprising administering to the subject a therapeutically effective amount of the composition of  claim 34 .  
     
     
         51 . A method of treating a subject having a disorder characterized by aberrant expression of a gene whose expression is regulated by P-TEFb, the method comprising administering to the subject a therapeutically effective amount of the nucleic acid molecules of any one of claims  24 .  
     
     
         52 . The method of any one of claims  44 - 51 , wherein the therapeutically effective amount is an amount effective to inhibit expression or activity of P-TEFb.  
     
     
         53 . The method of  claim 50  or  51 , wherein the gene is selected from the list of genes in Table 1.  
     
     
         54 . A method of treating a subject having a disorder characterized by aberrant expression of P-TEFb, the method comprising administering to the subject a therapeutically effective amount of the composition of  claim 54 .  
     
     
         55 . A method of treating a subject having a disorder characterized by aberrant expression of P-TEFb, the method comprising administering to the subject a therapeutically effective amount of the nucleic acid molecules of any one of claims  1 - 24 .  
     
     
         56 . The method of  claim 54  or  55 , wherein the therapeutically effective amount is an amount effective to inhibit expression or activity of P-TEFb.  
     
     
         57 . The method of any one of claims  50 - 56 , wherein the disorder is HIV/AIDS.  
     
     
         58 . The method of any one of claims  50 - 56 , wherein the disorder is cancer.  
     
     
         59 . The method of  claim 58 , wherein the cancer is selected from the group consisting of carcinomas, sarcomas, metastatic disorders and hematopoietic neoplastic disorders.  
     
     
         60 . The method of  claim 59  wherein the hematopoietic neoplastic disorder is a leukemia.  
     
     
         61 . A method for inhibiting unwanted cellular proliferation in a subject, the method comprising administering an effective amount of an inhibitor of CDK9.  
     
     
         62 . A method for inhibiting viral replication in a subject, comprising administering an effective amount of an inhibitor of CDK9.  
     
     
         63 . The method of  claim 61  or  62 , wherein the inhibitor of CDK9 reduces CDK9 expression.  
     
     
         64 . The method of  claim 63 , wherein the inhibitor is an antisense strand or a siRNA that is targeted to bind to a nucleic acid that encodes CDK9.  
     
     
         65 . The method of  claim 61  or  62 , wherein the inhibitor of CDK9 reduces P-TEFb activity.  
     
     
         66 . The method of  claim 65 , wherein the inhibitor is a small molecule, a dominant negative form of CDK9 or CycT1.  
     
     
         67 . The method of  claim 61 , wherein the unwanted cellular proliferation is cancer.  
     
     
         68 . The method of  claim 67 , wherein the cancer is selected from the group consisting of carcinomas, sarcomas, metastatic disorders and hematopoietic neoplastic disorders.  
     
     
         69 . The method of  claim 68 , wherein the hematopoietic neoplastic disorder is a leukemia.  
     
     
         70 . A method of treating a subject having a disorder characterized by aberrant expression of a gene whose expression is regulated by DSIF or Spt5, the method comprising administering to the subject a therapeutically effective amount of the composition of  claim 34 .  
     
     
         71 . A method of treating a subject having a disorder characterized by aberrant expression of a gene whose expression is regulated by DSIF or Spt5, the method comprising administering to the subject a therapeutically effective amount of the nucleic acid molecules of any one of claims  1 - 24 .  
     
     
         72 . The method of  claim 71  or  72 , wherein the gene is selected from the list of genes in Table 1.  
     
     
         73 . A method of treating a subject having a disorder characterized by aberrant expression of DSIF or Spt5, the method comprising administering to the subject a therapeutically effective amount of the composition of  claim 34 .  
     
     
         74 . A method of treating a subject having a disorder characterized by aberrant expression of DSIF or Spt5, the method comprising administering to the subject a therapeutically effective amount of the nucleic acid molecule of one of claims  1 - 24 .  
     
     
         75 . The method of any one of claims  70 - 74 , wherein the disorder is HIV/AIDS.  
     
     
         76 . The method of any one of claims  70 - 74 , wherein the disorder is cancer.  
     
     
         77 . The method of  claim 76  wherein the cancer is selected from the group consisting of carcinomas, sarcomas, metastatic disorders and hematopoietic neoplastic disorders.  
     
     
         78 . The method of  claim 77  wherein the hematopoietic neoplastic disorder is a leukemia.  
     
     
         79 . A method for inhibiting unwanted cellular proliferation in a subject, comprising administering an effective amount of an inhibitor of Spt5.  
     
     
         80 . A method for inhibiting viral replication in a subject, comprising administering an effective amount of an inhibitor of Spt5.  
     
     
         81 . The method of  claim 79  or  80 , wherein the inhibitor of Spt5 reduces Spt5 expression.  
     
     
         82 . The method of  claim 81 , wherein the inhibitor is antisense or a siRNA.  
     
     
         83 . The method of  claim 79  or  80 , wherein the inhibitor of Spt5 reduces DSIF activity.  
     
     
         84 . The method of  claim 83 , wherein the inhibitor is a small molecule, a dominant negative form of Spt5.  
     
     
         85 . The method of  claim 79  wherein the unwanted cellular proliferation is cancer.  
     
     
         86 . The method of  claim 85  wherein the cancer is selected from the group consisting of carcinomas, sarcomas, metastatic disorders and hematopoietic neoplastic disorders.  
     
     
         87 . The method of  claim 86  wherein the hematopoietic neoplastic disorder is a leukemia.